PROJECT SUMMARY/ABSTRACT. Pseudomonas aeruginosa, a Gram-negative bacterium, is an important opportunistic pathogen and the leading cause of chronic life-threatening lung infections in cystic fibrosis (CF) patients. P. aeruginosa is naturally antibiotic resistant and infections with this bacterium are notoriously difficult to treat. About 70% of CF patients are infected with P. aeruginosa by age 18 and the mean life expectancy is less than 40 years. A better understanding of how this bacterium evolves to cause these chronic respiratory infections is needed. Numerous phenotypic changes take place as P. aeruginosa transitions from the environment to causing life-shortening chronic CF lung infections. The emergence of isolates with a mucoid phenotype, due to the overproduction of the exopolysaccharide alginate is the most conspicuous; the isolation of mucoid strains is a prognosticator of clinical decline in these patients. Mucoid strains also have an additional less obvious polysaccharide alteration: these strains are generally lack lipopolysaccharide (LPS) O antigen, a phenotype referred to as ?LPS-rough?. Interestingly, this same conversion to a mucoid, LPS-rough form is prominent in P. aeruginosa isolates from patients with non-CF chronic bronchiectasis. In rare mucoid strains that do express LPS O antigen, different lengths of LPS O antigen are expressed compared to non-mucoid LPS-smooth strains: while LPS isolated from non-mucoid strains includes both long and very long O antigen, LPS isolated from mucoid strains shows decreased expression of very long O antigen but the same expression of long O antigen, which we suggest represents a transition state between the initial- and chronic-infecting forms. The goal of this 2-year Exploratory/Developmental Research Grant is to determine how mucoidy and loss of O antigen (as well as other genotypes) emerge during chronic colonization and how the bacteria and host respond to these infections. In Specific Aim 1, we will utilize the laboratory strain PAO1 and its mucoid derivative, PDO300, in a newly described murine model of chronic respiratory colonization and will follow the phenotypes and genotypes of the strains emerging and evolving during colonization as well as the bacterial and host response to infection. In Specific Aim 2, we will take a molecular approach to identify the genetic regulatory mechanisms controlling very long O antigen in a mucoid LPS-smooth strain and assess how alginate represses very long O antigen. The discovery of factors involved in the repression wzz2 (the gene which encodes the very long O antigen chain length regulator) will provide the relevant targets for therapeutic intervention. If we can inhibit the repression wzz2, this may set up a situation where the bacteria cannot maintain the mucoid phenotype and therefore would not be shielded from the innate host immune response or evolve into a chronic-colonizing form. The long-term goal of these studies will be to identify bacterial factors that can be targeted to inhibit chronic lung infection and host factors that can increase bacterial clearance and therefore improve the lives of CF patients.